If DNA could adopt only the structure of DNA with which we are most familiar - the canonical B-form double helix - it would be nearly devoid of biologic function. The ability of proteins to coerce DNA into a variety of non-canonical structures is an absolute prerequisite for many of the most important processing events that take place on the genome, including replication, transcription initiation, DNA repair, recombination, supercoiling, and packaging into chromatin. Although most of these processes are well-known at a descriptive level, much less is known about the pathways leading to DNA deformation and the associated energetic costs. The long-term goals of this research are to understand how the structure of DNA responds to torsional stress, and to elucidate the mechanisms by which proteins bring about alterations of DNA structure. The proposed studies will address this issue by trapping proteins in the act of inducing DNA distortion. A new and powerful technology that relies on the thermodynamically-driven formation of a disulfide crosslink between proteins and DNA will be employed to freeze otherwise transient complexes, thereby enabling high-resolution structural studies. The specific systems chosen for study are: RAG recombinase. This enzyme catalyzes a spectacular rearrangement of backbone connectivity in DNA, which is essential for the generation of immunologic diversity. Chemical and structural methods will be used to study the recombinase reaction mechanism in detail. Uracil DNA glycosylase. This enzyme initiates the repair of mutagenic uracil residues in DNA. How the enzyme locates these residues amidst the vast excess of normal DNA is a subject of the proposed investigation. HMGI-(Y). This protein stimulates the biological and biochemical activities of numerous diverse transcription factors and DNA- modifying enzymes. The mechanisms of stimulation is believed to involve manipulation of DNA architecture. The proposed experiments aim to resolve key issues regarding the DNA-binding mode of HMG-I(Y) and the mechanism by which it stimulates transactivation of the interferon-beta promoter by NF-kappaB.